The long term goal of this work is to understand, in precise molecular terms, how cell-type specific genes in budding yeast are transcriptionally repressed and to apply the principles developed from these studies to more complex organisms. The proposal can be divided into three basic questions: (l) How do pairs of homeodomain proteins (exemplified by alpha1 and alpha2) recognize with high affinity and specificity target DNA sequences? (2) What is the mechanism of action of a general transcriptional repression system (SSN6/TUP1) in yeast? (3) Are the components of the repression machinery and the mechanisms of repression conserved in more complex organisms? The experimental approaches emphasize the use of purified proteins and biochemical experiments to deduce molecular mechanisms. Genetic approaches in yeast are utilized (I) to test models developed from the biochemical studies and (2) to identify missing components of the repression system. Finally, complementation for function in yeast and PCR-based strategies are utilized to identify and analyze components of the repression systems in other organisms. Given the high degree of conservation of gene regulatory proteins and general transcription factors among all eucaryotes, it seems likely that many of the principles developed for the yeast proteins covered in this proposal will apply in other settings. A basic understanding of the molecular events underlying cell specialization provides not only a framework for understanding how the process can fail, but also provides the substrates and knowledge to design therapeutic strategies based on intervention.